PROJECT SUMMARY The use of antibody reagents with conformational selectivity and targeting well-defined epitopes is of paramount importance in elucidating detailed pathogenic mechanisms across biomedical science. Conversely, poorly characterized antibodies have been blamed for the lack of reproducibility of biomedical research studies. In the eye disorder glaucoma, dysfunction of the trabecular meshwork (TM) is linked closely to ocular hypertension, the causative risk factor for glaucoma, and the presence of the highly expressed protein myocilin, is used to validate relevant cells and eye tissues in research. However, the myocilin-directed antibodies currently in use have significant drawbacks for long term storage and fidelity, as well as for gleaning molecular insights, because myocilin is a modular protein prone to cleavage and misfolding. The availability of well-validated recombinant antibodies will be transformative for the vision research community. First, they will serve to standardize protocols across laboratories. Second, they will help illuminate myocilin explicit biological function/binding partners, and pathological changes to myocilin generally in glaucoma, which remain largely unknown. Third, they will help clarify why inherited mutations in myocilin localized to its olfactomedin domain (mOLF), are causative for open angle glaucoma. Finally, in the long term, myocilin-directed antibodies are likely adaptable to a new diagnostic or therapeutic for glaucoma. The objective of this proposal is to select and validate antibodies which are cross-reactive to mouse and human myocilin (85% identity) and bind specific conformational epitopes in the N-terminal (two coiled-coils) or C-terminal mOLF domains. Leveraging the long-standing collaboration between Georgia Tech?s Lieberman lab (molecular characterization of myocilin) and UT Austin?s Maynard Lab (therapeutic antibody engineering), an antibody recognizing a native N-terminal epitope has already been identified and validated. We now propose to identify additional conformational antibodies targeting other myocilin regions, motivated by the knowledge that myocilin is cleaved in cell culture and that epitopes may be occluded in when myocilin is in TM. Antibodies will be tested for conformational specificity by comparing detection in ELISA, dot, and Western blots, then optimized for stability and solubility, and epitopes delineated. Finally, antibodies will be tested for their ability to affinity purify myocilin from primary human TM cell culture, and via collaboration, to detect myocilin in mouse eye tissues. The expected outcomes are ~6 antibodies that recognize folded human and mouse myocilin domains and meet benchmarks for epitope recognition, biophysical properties, and research application. Broad dissemination of these reagents will enable an improved understanding of myocilin in a biological and pathological context, improve our overall comprehension of TM function, lead to the identification of new targets for anti-glaucoma therapies, as well as new glaucoma diagnostics and therapeutics.